A non-dicer RNase III and four other novel factors required for RNAi-Mediated transposon suppression in the human pathogenic yeast cryptococcus neoformans

Jordan E. Burke, Adam D. Longhurst, Prashanthi Natarajan, Beiduo Rao, John Liu, Jade Sales-Lee, Yasaman Mortensen, James J. Moresco, Jolene K. Diedrich, John R. Yates, Hiten D. Madhani

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. The RDE genes do not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies suggest that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endosiRNA biogenesis and transposon mobilization suppression in C. neoformans.

Original languageEnglish (US)
Pages (from-to)2235-2244
Number of pages10
JournalG3: Genes, Genomes, Genetics
Volume9
Issue number7
DOIs
StatePublished - Jul 1 2019

Keywords

  • RNA surveillance
  • RNAi transposon

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Genetics(clinical)

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    Burke, J. E., Longhurst, A. D., Natarajan, P., Rao, B., Liu, J., Sales-Lee, J., Mortensen, Y., Moresco, J. J., Diedrich, J. K., Yates, J. R., & Madhani, H. D. (2019). A non-dicer RNase III and four other novel factors required for RNAi-Mediated transposon suppression in the human pathogenic yeast cryptococcus neoformans. G3: Genes, Genomes, Genetics, 9(7), 2235-2244. https://doi.org/10.1534/g3.119.400330